Human interleukin-12 (IL-12) is a cytokine with a unique structure and pleiotropic effects (Kobayashi, et al. (1989) J Exp Med 170:827-845; Seder, et al. (1993) Proc. Natl. Acad. Sci. 90:10188-10192, Ling, et al. (1995) J Exp Med 154:116-127; Podlaski, et al. (1992) Arch. Biochem. Biophys. 294:230-237). IL-12 plays a critical role in the pathology associated with several diseases involving immune and inflammatory responses. A review of IL-12, its biological activities, and its role in disease can be found in Trinchieri, G. (2003) Nat. Rev. Immun. 3:133-146. Structurally, IL-12 is a heterodimeric protein (referred to as the “p70 protein”) comprising a 35 kDa subunit (p35) and a 40 kDa subunit (p40) which are linked together by a disulfide bridge. The heterodimeric protein is produced primarily by antigen-presenting cells such as monocytes, macrophages and dendritic cells. These cell types also secrete an excess of the p40 subunit relative to p70 subunit. The p40 and p35 subunits are genetically unrelated and neither has been reported to possess biological activity, although the p40 homodimer may function as an IL-12 antagonist.
Functionally, IL-12 plays a central role in regulating the balance between antigen-specific T helper type 1 (Th1) and type 2 (Th2) lymphocytes. The Th1 and Th2 cells govern the initiation and progression of autoimmune disorders, and IL-12 is critical in the regulation of Th1-lymphocyte differentiation and maturation. Cytokines released by the Th1 cells are inflammatory and include interferon gamma (IFN-γ), IL-2, and lymphotoxin (LT). Th2 cells secrete IL-4, IL-5, IL-6, IL-10 and IL-13 to facilitate humoral immunity, allergic reactions, and immunosuppression. Consistent with the preponderance of Th1 responses in autoimmune diseases and the proinflammatory activities of IFN-γ, IL-12 may play a major role in the pathology associated with many autoimmune and inflammatory diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis (PS) and Crohn's disease (CD).
Human patients with MS have demonstrated an increase in IL-12 expression as documented by p40 mRNA levels in acute MS plaques (Windhagen et al., (1995) J Exp. Med. 182:1985-1996). In addition, ex vivo stimulation of antigen-presenting cells with CD40L expressing T cells from MS patients resulted in increased IL-12 production compared with control T cells, consistent with the observation that CD40/CD40L interactions are potent inducers of IL-12. Elevated levels of IL-12 p70 have been detected in the synovia of RA patients compared with healthy controls (Morita et al. (1998) Arth. and Rheumat. 41:306-314). Cytokine messenger ribonucleic acid (mRNA) expression profile in the RA synovia identified predominantly Th1 cytokines (Bucht et al. (1996) Clin. Exp. Immunol. 103:347-367). IL-12 also appears to play a critical role in the pathology associated with Crohn's disease. Increased expression of INF-γ and IL-12 has been observed in the intestinal mucosa of patients with this disease (Fais et al. (1994) J Interferon Res. 14:235-238; Parronchi et al. (1997) Am. J. Path. 150:823-832; Monteleone et al. (1997) Gastroent. 112:1169-1178, and Berrebi et al. (1998) Am. J Path 152:667-672). The cytokine secretion profile of T cells from the lamina propria of CD patients is characteristic of a predominantly Th1 response, including greatly elevated IFN-γ levels (Fuss, et al. (1996) J Immunol. 157:1261-1270). Moreover, colon tissue sections from CD patients show an abundance of IL-12 expressing macrophages and IFN-γ expressing T cells (Parronchi et al (1997) Am. J. Path. 150:823-832).
IL-23 is also a heterodimeric cytokine and belongs to a family of five such heterodimeric cytokines including IL-12 and IL-27 (Trinchieri et al., (2003) Immunity 19:641-644). IL-23 shares the identical p40 subunit as IL-12, but it is associated with a p19 subunit via a disulphide-linkage. The p19 subunit is structurally related to IL-6, granulocyte-colony stimulating factor (G-CSF), and the p35 subunit of IL-12. IL-23 is produced by similar cell types as IL-12, and its receptor is expressed on T cells, NK cells, and phagocytic and dendritic hematopoietic cells. IL-23 mediates signaling by binding to a heterodimeric receptor, comprised of IL-23R and IL-12beta1. The IL-12beta1 subunit is shared by the IL-12 receptor, which is composed of IL-12beta1 and IL-12beta2. IL-23 does share overlapping functions with IL-12 (by inducing IFN-γ production, Th1 cell differentiation and activating the antigen-presenting functions of dendritic cells) however it selectively induces proliferation of memory T cells (Oppmann et al. (2000) Immunity 13:715-725, Parham, et al. (2002) J. Immunol. 168:5699-5708).
The role of IL-23 in autoimmune inflammation has been dissected in part through studies with p19 knockout mice (Murphy et al. J Exp Med 198:1951-1957; Cua et al. (2003) Nature 421:744-748). Studies have demonstrated that IL-23 modulates immune response to infection (see, e.g., Pirhonen, et al. (2002) J. Immunol. 169:5673-5678; Broberg, et al. (2002) J. Interferon Cytokine Res. 22:641-651; Elkins, et al. (2002) Infection Immunity 70:1936-1948; Cooper, et al. (2002) J. Immunol. 168:1322-1327). IL-23 is thought to play a role in immune-mediated inflammatory diseases (Langrish et. al. (2004) Immunological Reviews 202: 96-105).
Due to the role of human IL-12 in a variety of human disorders, therapeutic strategies have been designed to inhibit or counteract IL-12 activity. In particular, antibodies that bind to, and neutralize, IL-12 have been sought as a means to inhibit IL-12 activity. Some of the earliest antibodies were murine monoclonal antibodies (mAbs), secreted by hybridomas prepared from lymphocytes of mice immunized with IL-12 (see e.g., Strober et al., PCT Publication No. WO 97/15327; Gately et al., WO9937682 A2; Neurath et al., J Exp. Med 182:1281-1290 (1995); Duchmarm et al., J Immunol. 26:934-938 (1996)). These murine IL-12 antibodies are limited for their use in vivo due to problems associated with administration of mouse antibodies to humans, such as short serum half life, an inability to trigger certain human effector functions and elicitation of an unwanted immune response against the mouse antibody in a human (the “human anti-mouse antibody” (HAMA) reaction).
One approach to overcome the problem problems associated with use of fully murine antibodies in humans is to generate fully human antibodies such as those disclosed in Salfeld et al., PCT publication No. WO 00/56772 A1. Other approaches to overcome the problems associated with use of fully murine antibodies in humans have involved genetically engineering the antibodies to be more “human-like.” For example, chimeric antibodies, in which the variable regions of the antibody chains are murine-derived and the constant regions of the antibody chains are human-derived, have been prepared (Junghans, et al. (1990) Cancer Res. 50:1495-1502; Brown et al. (1991) Proc. Natl. Acad. Sci. 88:2663-2667; Kettleborough et al. (1991) Prot. Engineer. 4:773-783). Such chimeric antibodies to IL-12 are also disclosed in Peritt et al. PCT publication No. WO2002097048A2. However, because these chimeric antibodies still retain murine variable chain sequences, they still may elicit an unwanted immune reaction, the human anti-chimeric antibody (HACA) reaction especially when administered for prolonged periods.
There is a need in the art for improved antibodies capable of binding the p40 subunit of IL-12 (IL-12p40). Preferably the antibodies bind IL-12 and/or IL-23. Preferably the antibodies are capable of neutralizing IL-12 and/or IL-23. The present invention provides a novel family of binding proteins, CDR grafted antibodies, humanized antibodies, and fragments thereof, capable binding IL-12p40, binding with high affinity, and binding and neutralizing IL-12 and/or IL-23.